The perforant path (PP) undergoes synaptic changes in the course of aging and dementia. Previous studies attempting to assess the integrity of the PP in humans using diffusion tensor imaging (DTI) were limited by low resolution and the inability to identify PP fibers specifically. Here we present an application of DTI at ultrahigh submillimeter resolution that has allowed us to successfully identify diffusion signals unique to the PP and compare the intensity of these signals in a sample of young adults and older adults. We report direct evidence of age-related PP degradation in humans in vivo. We find no evidence of such loss in a control pathway, the alveus, suggesting that these findings are not evidence for a global decline. We also find no evidence for specific entorhinal gray matter atrophy. The extent of PP degradation correlated with performance on a word-list learning task sensitive to hippocampal deficits. We also show evidence for gray matter diffusion signals consistent with pyramidal dendrite orientation in the hippocampus and cerebral cortex. Ultrahigh-resolution microstructural DTI is a unique biomarker that can be used in combination with traditional structural and functional neuroimaging methods to enhance detection of Alzheimer disease in its earliest stages, test the effectiveness of new therapies, and monitor disease progression.T here is converging evidence that one of the earliest locations in the brain to undergo age-related change is the medial temporal lobe (MTL) region (1, 2), which plays an important role in learning new facts and remembering events (3, 4). Selective structural and functional changes in the MTL are evident with aging and specifically target white matter connectivity and cellular function (1). One MTL pathway that is particularly vulnerable to age-related changes is the perforant path (PP) (5-7). This pathway perforates the subiculum carrying input from the entorhinal cortex to the hippocampal formation ( Fig. 1 A and B). The integrity of this pathway is essential for normal hippocampal function (8). Studies in rodents have shown that the PP input to the hippocampus is reduced by approximately one third in aged rats compared with young rats (9, 10) and that stimulation of the PP in aged rats results in less excitation than in young rats (1). Histological studies have observed reduced myelin staining in the PP in Alzheimer disease (AD) (8), and more recently, synaptic loss in this region in individuals with mild cognitive impairment (11). Thus, numerous studies have suggested that the PP undergoes changes with aging and in the course of dementia. However, observing evidence of this degradation in humans in vivo, a key requirement of any viable biomarker, has remained elusive.Diffusion tensor imaging (DTI) is used to investigate the microstructural features of white matter (12), and has recently been applied to the study of aging and dementia (13). DTI is based on the principle that water will diffuse more readily along the principal axis of an axon than perpendicular to ...